Improved survival of SARS COV-2-infected K18- hACE2 mice treated with adenosine A2AR agonist

Abstract

A life-threatening manifestation of Covid-19 infection is a cytokine storm that requires hospitalization and supplemental oxygen. Various strategies to reduce inflammatory cytokines have had some success in limiting cytokine storm and improving survival. Agonists of adenosine A_2A receptors (A_2AR) reduce cytokine release from most immune cells. Apadenoson is a potent and selective anti-inflammatory adenosine analog that reduces inflammation. When administered by subcutaneous osmotic pumps to mice infected with SARS CoV-2, Apadenoson was found to improve the outcomes of infection as measured by a decrease in weight loss, improved clinical symptoms, reduced levels of proinflammatory cytokines and chemokines in bronchial lavage (BAL) fluid, and enhanced survival of K18-hACE2 transgenic mice. These results support further examination of A_2AR agonists as therapies for treating cytokine storm due to COVID-19.

Keywords: Adenosine agonists; Apadenoson; COVID-19; SARS CoV2; Therapy.

Fig. 1

Treatment with Apadenoson improved survival and delayed time to death. Mice were treated prophylactically, 18 h before, or therapeutically, 5 h after infection, with Apadenoson delivered by an osmotic pump. Data combined from three independent experiments. A) Survival curves, used Log Rank (Mantel Cox) test, p = 0.0002*** for drug with delay compared to vehicle, B) Average weight loss: P values from linear mixed model for difference in slope of vehicle compared to drug, p = 0.02*, vehicle compared to drug with delay, p = 0.002** and C) Clinical Scores: P values from linear mixed model for difference in slope of vehicle compared to drug, p = 0.027*, vehicle compared to drug with delay, p = 0.001**. The linear mixed model analysis accounts for survivor bias. Mice were examined twice daily for clinical symptoms and scored using the following criteria: weight loss (0–5, in 5% of initial weight loss increments), reduced activity (0–3), ruffled fur appearance and hunched posture (0–2), and an eye closure (0–2). Mice were euthanized when weight was <80% of initial weight or when they scored a maximum in two symptom categories. These results are combined from three independent experiments with a total of 18 mice in the vehicle treatment group and 17 mice each in the drug and drug with delay treatment groups.

Fig. 2

Weight loss (A) and clinical scores (B) of SARS CoV-2 infected mice treated with Apadenoson (drug with delay) or saline (vehicle) were not statistically different on day five post-infection. Mean ± SD is shown. Statistically evaluated using unpaired t-test. These results are combined from two independent experiments.

Fig. 3

Cytokine and chemokine levels in SARS CoV-2-infected mice treated with Apadenoson are suppressed. Mice were euthanized on day five post-infection. Cytokine/chemokine levels were measured in BAL fluid by a Luminex. Combined results of two independent experiments are presented. Statistical differences in cytokine/chemokine levels were analyzed using Welch's ANOVA, followed by Dunnett's T3 multiple comparisons. Shown p values are from the Dunnett's Test with raw p values < 0.05*, 0.005**.

Fig. 4

Less Viral burden on day five post-infection in mice treated therapeutically with Apadenoson. A) Lungs were stained with H&E or a SARS CoV-2 anti-NP antibody. Representative mice are shown. Mice #2 and 6 were vehicle-treated; Mice #8 and #11 were therapeutically (drug with delay, 5 h after infection) -treated. H&E-stained lungs are shown at a higher magnification than IHC slides. B) Percentage of viral staining in the lungs by treatment group. P value = 0.0298, unpaired t-test. The percent viral burden in the lungs was determined using ImageJ. These results are combined from two independent experiments.

Fig. 5

Viral titers and burdens in lungs of surviving Apadenoson-treated mice on day 12 post-infection. A) Viral lung burden was significantly reduced in mice treated therapeutically with Apadenoson after infection compared to both vehicle (p = 0.0014) and with prophylactic treatment with drug prior to infection (p = 0.043), Dunnett's T3 multiple comparisons B) Viral titers of lung homogenates trended lower in drug with delay treated mice but were not significant using Welch's ANOVA. Undetectable viral titers were assigned a value of 1. These results are combined from two independent experiments. Lungs were harvested on day 12 post-infection. The viral burdens and titers in vehicle treated mice were determined on day 5 post-infection, the day they met the criteria for euthanasia. See Supplemental Fig. 2 for IHC images.

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